The flow cytometry myeloid progenitor count: A reproducible parameter for diagnosis and prognosis of myelodysplastic syndromes.

BACKGROUND: The bone marrow blast count is central to the diagnosis and monitoring of myelodysplastic syndromes (MDS). It is an independent risk factor for worse prognosis whether based on the morphology blast count or the flow cytometry (FC) myeloid progenitor (MyP) count. It is a principal population in FC MDS analysis also because once defined; it provides significant contributions to the overall FC MDS score. METHODS: We elected to investigate inter-analyst agreement for the most fundamental parameter of the FC MDS diagnostic score: the MyP count. A common gating strategy was agreed and used by seven cytometrists for blind analysis of 34 routine bone marrows sent for MDS work-up. Additionally, we compared the results with a computational approach. RESULTS: Concordance was excellent: Intraclass correlation was 0.993 whether measuring %MyP of total cells or CD45+ cells, and no significant difference was observed between files from different centers or for samples with abnormal MyP phenotypes. Computational and manual results were similar. Applying the common strategy to individual laboratories' control cohorts produced similar MyP reference ranges across centers. CONCLUSION: The FC MyP count offers a reliable diagnostic and prognostic measurement in MDS. The use of manual and computational approaches side by side may allow for optimizing both strategies. Considering its known prognostic power, the MyP count could be considered a useful and reliable addition to existing prognostic scoring systems.

Reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry: An European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) Harmonisation project.

The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the role of new markers. The aim of this project was to identify reproducible criteria and consensus on markers recommended for the diagnosis of CLL. ERIC/ESCCA members classified 14 of 35 potential markers as "required" or "recommended" for CLL diagnosis, consensus being defined as >75% and >50% agreement, respectively. An approach to validate "required" markers using normal peripheral blood was developed. Responses were received from 150 participants with a diagnostic workload >20 CLL cases per week in 23/150 (15%), 5-20 in 82/150 (55%), and <5 cases per week in 45/150 (30%). The consensus for "required" diagnostic markers included: CD19, CD5, CD20, CD23, Kappa, and Lambda. "Recommended" markers potentially useful for differential diagnosis were: CD43, CD79b, CD81, CD200, CD10, and ROR1. Reproducible criteria for component reagents were assessed retrospectively in 14,643 cases from 13 different centers and showed >97% concordance with current approaches. A pilot study to validate staining quality was completed in 11 centers. Markers considered as "required" for the diagnosis of CLL by the participants in this study (CD19, CD5, CD20, CD23, Kappa, and Lambda) are consistent with current diagnostic criteria and practice. Importantly, a reproducible approach to validate and apply these markers in individual laboratories has been identified. Finally, a consensus "recommended" panel of markers to refine diagnosis in borderline cases (CD43, CD79b, CD81, CD200, CD10, and ROR1) has been defined and will be prospectively evaluated. © 2017 International Clinical Cytometry Society.

Genetics and Prognostication in Splenic Marginal Zone Lymphoma: Revelations from Deep Sequencing.

PURPOSE: Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies. EXPERIMENTAL DESIGN: We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted resequencing and explored potential clinical implications in a multinational cohort of 175 patients with SMZL. RESULTS: We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%), and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B-cell receptor immunoglobulins, and were associated with a short median time to first treatment (0.12 vs. 1.11 years; P = 0.01). In multivariate analysis, mutations in NOTCH2 [HR, 2.12; 95% confidence interval (CI), 1.02-4.4; P = 0.044] and 100% germline IGHV gene identity (HR, 2.19; 95% CI, 1.05-4.55; P = 0.036) were independent markers of short time to first treatment, whereas TP53 mutations were an independent marker of short overall survival (HR, 2.36; 95 % CI, 1.08-5.2; P = 0.03). CONCLUSIONS: We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively.

Clonal B-cell lymphocytosis exhibiting immunophenotypic features consistent with a marginal-zone origin: is this a distinct entity?

The biological and clinical significance of a clonal B-cell lymphocytosis with an immunophenotype consistent with marginal-zone origin (CBL-MZ) is poorly understood. We retrospectively evaluated 102 such cases with no clinical evidence to suggest a concurrent MZ lymphoma. Immunophenotyping revealed a clonal B-cell population with Matutes score ≤2 in all cases; 19/102 were weakly CD5 positive and all 35 cases tested expressed CD49d. Bone marrow biopsy exhibited mostly mixed patterns of small B-lymphocytic infiltration. A total of 48/66 (72.7%) cases had an abnormal karyotype. Immunogenetics revealed overusage of the IGHV4-34 gene and somatic hypermutation in 71/79 (89.8%) IGHV-IGHD-IGHJ gene rearrangements. With a median follow-up of 5 years, 85 cases remain stable (group A), whereas 17 cases (group B) progressed, of whom 15 developed splenomegaly. The clonal B-cell count, degree of marrow infiltration, immunophenotypic, or immunogenetic findings at diagnosis did not distinguish between the 2 groups. However, deletions of chromosome 7q were confined to group A and complex karyotypes were more frequent in group B. Although CBL-MZ may antedate SMZL/SLLU, most cases remain stable over time. These cases, not readily classifiable within the World Heath Organization classification, raise the possibility that CBL-MZ should be considered as a new provisional entity within the spectrum of clonal MZ disorders.